PROJECT SUMMARY/ABSTRACT Proper assembly and functioning of neuronal circuits in the neocortex, a brain structure critical for all higher- order functions, relies on the formation of specific synaptic connections between excitatory pyramidal neurons (PyNs) and different types of inhibitory GABAergic interneurons. One subset of interneurons that exerts powerful control over PyN spiking is the chandelier cell (ChC), which forms connections specifically at the site of action potential initiation in PyNs, referred to as the axon initial segment (AIS). Due to the unique connections formed between the terminals of ChC axonal arbors and the AISs of large populations of PyNs, ChCs are ideally suited to control the output of excitatory cortical networks. Hence, it is not surprising that ChC connectivity defects are linked to brain disorders, such as schizophrenia and autism spectrum disorder. Despite the importance of ChCs, the molecular mechanisms underlying their subcellular innervation of PyN AISs are unknown. To approach this, we initiated an in utero electroporation (IUE)-based in vivo RNAi screen of known neocortical PyN-expressed axonal cell adhesion molecules (CAMs) and select Ephs/ephrins. Strikingly, of all the molecules tested, we found the CAM L1CAM to be the only protein required for PyN AIS innervation by ChCs. In addition, based on single- ChC RNAseq data, we investigated the role of ChC-expressed netrin receptor Unc5b in ChC/PyN AIS innervation and found that it plays a key role in ChC axon terminal development and connectivity. Our findings provide a unique entry point for studies on the molecular basis of ChC/PyN connectivity. This application aims to elucidate how PyN L1CAM governs selective AIS innervation, to identify L1CAM?s presynaptic binding partner(s) on ChCs, and to scrutinize the mechanism of Unc5b in ChC axon terminal development and connectivity. To this end, Aim 1 will use molecular tools to disrupt interactions between L1CAM and the AIS cytoskeleton to assess whether cytoskeleton-mediated L1CAM clustering at the AIS is required for proper ChC/PyN AIS innervation. High resolution imaging will be performed to investigate the distribution of surface L1CAM on the AIS and distal axon. Also, inducible RNAi constructs delivered by IUE to temporally regulate PyN L1CAM levels in vivo will be used to determine whether PyN L1CAM is required for the establishment and/or maintenance of ChC/PyN innervation. Aim 2 will identify the presynaptic binding partner(s) of L1CAM on ChC terminals. Our preliminary data suggest that neuropilin-1 (Nrp1) is the L1CAM partner on ChCs required for ChC/PyN innervation. This will be tested by depleting Nrp1 in ChCs, using RNAi technology and conditional Nrp1 knockout mice. The Nrp1 domain(s) required for L1CAM binding will also be defined. Aim 3 will determine whether Unc5b governs ChC cartridge development by regulating terminal axon branching. We will also test whether the LARG/RhoA/ROCK pathway mediates Unc5b?s effect on ChC axon terminal development and connectivity using molecular/cellular tools and ChC-targeting IUE. Together, our studies will provide first insight into the mechanisms governing ChC/PyN connectivity and shed new light on the connectivity defects underlying common brain disorders. !